INTRODUCTION:

Alprazolam (Fig-1) is chemically, 8-chloro-1-methyl-6-phenyl-4H-(1,2,4) triazolo (4,3a)1,4-benzodiazepine, which is a short acting anxiolytic drug¹. Alprazolam is a triazobenzodiazepine agent with anxiolytic, sedative-hypnotic, anticonvulsant, amnesic and skeletal muscle relaxant properties². It has fast onset action and symptomatic relief. Alprazolam may be habit-forming, and long-term use and abuse may cause a physical dependence to develop along with withdrawal reactions during abrupt or rapid discontinuation^3,4. Although the side-effect profile of Alprazolam may occur in some patients and are more likely the higher the dosage taken. Some side-effects may disappear with continued treatment, if signs of an allergic reaction occur such as hives, difficulty in breathing, swelling of face, lips, tongue, or throat⁵.

Fig -1: Structure of Alprazolam

Alprazolam has been determined by voltammetry⁶, HPLC⁷, HPTLC-UV absorption densitometry⁸. RP-LC⁹, RP-HPLC¹⁰ and GC-MS^11,12. Determination of Alprazolam in serum by HPLC was repoted¹³. Ayaz Mohd et al reported UV-Absorption and fluorimetric methods for the determination of Alprazolam in pharmaceutical formulations14. A new HPLC analytical method for the determination of Alprazolam in tablets was also reported¹⁵. Potentiometric16 and LC-Tandem MS method¹⁷ for the determ inatioin of Alprazolam, method development, validation and determination of Alprazolam in its pharmaceutical dosage by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone are available in the literature¹⁸. Various spectrophotometric methods for the estimation of Alprazolam in tablet dosage form¹⁹, using ferric chloride and indigo carmine²⁰, by area under curve²¹ and by second and third order derivative methods²² are also available in the literature.

The chemical features of the Alprazolam drug molecule offers a lot of scope for the development of new methods for its determination with better sensitivity, specificity, precision and accuracy. The reported chromatographic techniques require expensive experimental set-up and are not affordable in every laboratories for routine analysis. The literature survey revealed that, although, UV-vis spectrophotometric methods for the determination of Alprazolam are available, a little attention was paid to the development of spectrophotometric methods for its determination using dyes. Spectrophotometry is considered as the most convenient analytical technique because of its inherent simplicity, low cost and wide availability in most quality control laboratories. Because of its physiological significance, the quantitative determination of Alprazolam attracted the attention of analytical chemists and almost all analytical methods have been applied to accomplish the purpose. However methods on spectrophotometric determination of this drug involving ion-pair complexes with common and versatile acidic dyes viz., bromothymol blue (BTB), bromophenol blue (BPB) and bromocresol green (BCG) are not reported yet. This prompted the authors to develop extractive spectrophotometric methods for the determination of Alprazolam using above mentioned dyes. In continuation of our study on the estimation of drugs using triphenyl methane dyes^23,24, we now report in this paper, three newly developed and validated spectrophotometric estimation methods of Alprazolam in bulk and pharmaceutical formulations using triphenyl methane dyes viz., BTB, BPB and BCG. The developed methods involve formation of coloured chloroform extractable ion-pair complexes of the drug with dyes in acidic medium. These methods are based on ion-pair complexation of drug with dyestuffs and subsequent extraction into chloroform and to measure the absorbance of colour complex. The proposed methods have the advantages of speed and simplicity besides being accurate and precise, and can be adopted by the pharmaceutical laboratories for industrial quantitative analysis.

EXPERIMENTAL:

Materials:

Alprazolam was procured from Srini Pharmaceuticals Limited, Hyderabad as a gift sample. The dyestuffs viz., BTB, BPB and BCG (AR grade) supplied by SD Fine Chemicals Ltd. Mumbai, were used without any further purification. The dyestuffs were used as 0.025% solutions in doubly distilled water. Sodium acetate-hydrochloric acid buffers of required pH were prepared by mixing 50ml of 1.0M sodium acetate solution with calculated volume of 1.0 M HCl solution and diluted to 250ml with doubly distilled water. The pH of each solution was adjusted to an appropriate value with the aid of a pH meter. Chloroform (HPLC grade) supplied by SD Fine Chemicals Ltd. Mumbai was used throughout the work.

Instruments:

The spectra of ion-pair complexes have been recorded on Elico double beam SL 210 spectrophotometer using quartz cells of 10mm path length. An Elico model Li-120 pH meter was used for pH measurement.

Methods:

Method A:

The basis for the method A is the interaction of the drug with dye, BTB to form chloroform extractable ion-pair complex which shows absorption around 410nm. Increase in the absorbance with the concentration of drug formed a basis for the quantification of the drug. 0.025% solutions of dye stuff in doubly distilled water and CH₃COONa-HCl buffer of pH 2.5 were used and the pH of the reaction mixture in each case was adjusted to required value with the help of a pH meter.

Method B:

The basis for the method B is the interaction of the drug with dye, BPB to form chloroform extractable ion-pair complex which shows absorption around 408nm. Increase in the absorbance with the concentration of drug formed a basis for the quantification of the drug by this method. The pH of the solution was maintained at 2.5 using CH₃COONa-HCl buffer. All the other experimental details are similar as mentioned in method A.

Method C:

The basis for the method C is the interaction of the drug with dye, BCG to form chloroform extractable ion-pair complex which shows absorption around 405nm. The increase in the absorbance with the concentration of drug formed the basis for the quantification of the drug. The pH of the solution was maintained at 2.5 using CH₃COONa-HCl buffer. All the other experimental details are similar as mentioned in method A.

RESULTS AND DISCUSSION:

Alprazolam forms ion-pair complexes in acidic buffer with dyestuffs such as bromothymol blue (BTB), bromophenol blue (BPB) and bromocresol green (BCG) and these complexes are quantitatively extracted into chloroform. Ion-pair complexes of drug with BTB, BPB and BCG absorbed maximally at 410, 408 and 405nm respectively (Fig. 2a, 2b and 2c). The reagent blank under similar conditions showed no absorption.

Fig-2: Absorption spectra of Alprazolam-dye complex extracted into 10 ml chloroform

a. drug = 25.0mg ml^-1 + 5 ml of 0.025% BTB + 5 ml of pH 2.5 buffer

b. drug = 22.5mg ml^-1 + 5 ml of 0.025% BPB + 5 ml of pH 2.5 buffer

c. drug = 20.0mg ml^-1 + 5 ml of 0.025% BCG + 5 ml of pH 2.5 buffer

Alprazolam is a benzodiazepine derivative. The nitrogen of diazepine ring at fourth position is protonated in acid medium, while sulphonic acid group present in BTB, BPB and BCG undergoes dissociation in the pH range 1-5. The colour of such dyes is due to the opening of lactoid ring and subsequent formation of quinoid group. It is supposed that the two tautomers are present in equilibrium but due to strong acidic nature of the sulphonic acid group, the quinoid body must predominate. Finally the protonated Alprazolam forms ion-pairs with the dyestuffs which are quantitatively extracted into chloroform. The possible reaction mechanism is proposed and given in Scheme 1.

Scheme – 1: Alprazolam-dye ion pair complex

Bromothymol blue : R₁ = isopropyl, R₂ = -CH₃

Bromothymol blue : R₁ = -Br, R₂ = -H

Bromocresol green : R₁ = -Br, R2 = -CH₃

Calibration curve:

Different aliquots of drug solution were transferred into 125ml separating funnel. To this 5ml of buffer, 5ml of dye were added and total volume was made up to 20ml with water. 10ml of chloroform was added and the contents were shaken for 5 min. The two layers were allowed to separate for 5 min. The organic layer was separated and absorbance of yellow colored solution which is stable at least for 3 hrs is measured at 420 nm against blank similarly prepared. The same procedure of analysis is followed either for assay of pure drug or for dosage form. The calibration graphs (Fig-3) are linear over the concentration ranges and are within the permissible range. The optical characteristics and statistical data for the regression equation of the proposed methods are presented in Table 1.

Fig-3: Calibration graphs for Alprazolam-BTB, BPB and BCG Ion-pair complexes

Table-1: Optical characteristics and statistical analysis for the regression equation of the proposed methods for the estimation of Alprazolam

Parameters	Extraction methods with^b
Parameters	BTB	BPB	BCG
λ_max (nm)	410	408	405
Beer’s law limit (μg ml^-1)	3.0-25	4.0-30	4.5-40
Molar absorptivity (L mol^-1 cm^-1)	11839	13377	13966
Formation constant, K, M^-1	1.51x10⁶	1.72x10⁶	1.86x10⁶
Sandell sensitivity (μg cm^-2)	0.02602	0.02302	0.02192
Slope (specific absorptivity), b	0.03844	0.04343	0.04563
Intercept (a)	-0.0038	0.00523	0.05688
Correlation coefficient (r)	0.99942	0.99998	0.99981
Standard deviation of intercepts (% n=6)	0.00276	0.00321	0.00422
Limit of detection, μgml^-1	0.2367	0.2439	0.3045
Limit of quantification, μgml^-1	0.7174	0.7391	0.9227
Regression equation^a	Y=0.03844C± 0.0038	Y=0.04343C± 0.00523	Y=0.04563C± 0.05688

^aWith respect to Y=bc+a, where C is the concentration (μg ml^-1) and Y is absorbance

^bSix replicate samples

Procedure for the assay of pure drug:

Five different solutions of pure drug in the range of calibration curve were selected and the recovery experiments were performed. The recoveries and their relative standard deviations are tabulated in Table 2.

Table-2: Application of proposed methods for the analysis of Alprazolam in pure form

Taken (μg ml^-1)	Proposed methods						Reference method Recovery (%)
Taken (μg ml^-1)	Found (μg ml^-1)			Recovery (%)
	BTB	BPB	BCG	BTB	BPB	BCG
8	8.07	8.12	7.95	100.87	101.50	99.37	98.74
12	11.98	12.12	11.95	99.83	101.00	99.58	101.12
16	16.22	15.95	16.12	101.37	99.68	100.75	101.98
20	19.88	19.98	20.24	99.40	99.90	101.20	101.12
24	23.84	24.15	24.22	99.33	100.62	100.92	100.32
							99.64
							100.58
							101.56
RSD (%)				0.9128	0.7524	0.8276	1.0502
Mean±SD				100.16 ± 0.914	100.54 ± 0.756	100.36 ± 0.830	100.63 ± 1.0568
t-test				0.2504	0.5672	0.4135
F-test				0.2815	0.5453	0.4279

Table-3: Application of proposed methods for the analysis of Alprazolzm in pharmaceutical form

Taken (μg ml^-1) Alpram 0.5mg	Proposed methods						Reference method Recovery (%)
	Found (μg ml^-1)			Recovery (%)
	BTB	BPB	BCG	BTB	BPB	BCG
8	7.88	7.92	8.16	98.50	99.00	102.00	101.42
12	12.12	11.96	12.08	101.00	99.66	100.66	99.88
16	15.95	16.22	15.94	99.68	101.37	99.62	99.42
20	20.08	30.08	19.98	100.40	100.40	99.90	99.12
24	24.22	23.92	23.92	100.91	99.66	99.66	101.02
							101.62
							99.32
							99.22
RSD (%)				1.0349	0.9035	0.9998	1.0506
Mean±SD				100.09 ± 1.0359	100.02 ± 0.9036	100.36 ± 1.0035	100.13 ± 1.052
t-test				0.02672	0.26214	0.0817
F-test				0.0336	0.2902	0.0997

Procedure for the assay of dosage forms:

Ten tablets of Alpram 0.5mg each are powdered and dissolved in doubly distilled water and stirred thoroughly, filtered through a Whatman No. 42 filter paper. This solution was transferred into 100ml standard volumetric flask and diluted with doubly distilled water as required. Different solutions of drug in the range of calibration curve were chosen and the assay was estimated using the calibration curve. The results of the recovery experiments are tabulated in Table 3.

Stoichiometry:

In order to establish molar ratio between Alprazolam and dyestuffs used, the Job’s method of continuous variation²⁵ has been applied. In this method, solutions of drug and dyestuff with identical molar concentrations (8 x 10^-5M) were mixed in varying volume ratios in such a way that the total volume of each mixture was the same. The absorbance of each solution was measured and plotted against the mole fraction of the drug, [drug]/ [drug] + [dyestuff] (Fig-4). This measurement showed that 1:1 complex was formed with each dyestuff. The formation constants²⁶ were also estimated and found to be 1.51x10⁶, 1.72x10⁶ and 1.86x10⁶K M^-1for complexes with BTB, BPB and BCG respectively.

Fig-4: Continuous-variation study of drug-dye systems [Alprazolam] = [Dye] = 8x10^-5M

Optimization of the factors affecting the absorbance:

The influence of pH on the ion-pair formation of Alprazolam with various dyestuffs has been studied using sodium acetate-hydrochloric acid buffer. The results are shown in Fig-5. It is evident that absorbance of complexes with BTB, BPB and BCG was found to be constant within the pH ranges 2.2-3.3, 2.0-3.0 and 2.5-3.8 respectively. Thus, all the absorbance measurements were made at pH 2.5, 2.8, 3.5 with BTB, BPB and BCG respectively.

Fig-5: Effect of pH [Alprazolam] = [8µg ml^-1], [Dye] = 5ml of 0.025%

The effect of dyestuff concentrations was also studied by adding different volumes of dyestuff to a constant amount of Alprazolam (8µg ml^-1). It is apparent from Fig-6 that the maximum absorbance, in each case, was found with 3.0ml of dyestuff, beyond which absorbance was constant. Thus, 5ml of each dyestuff was used for ion-pair formation throughout the experiment.

Fig-6: Influence of the volume of 0.025% Dye [Alprazolam] = [8µg ml^-1]

A systematic study of the effect of foreign species present along with Alprazolam on the determination of Alprazolam at 8µg ml^-1 levels was undertaken. This study was carried out by following the proposed procedures for a 10ml sample system, by adding a known amount of foreign species to an Alprazolam solution of 8µg ml^-1. Table 4 summarizes the results obtained. However, the drug content from the powdered capsules was extracted into chloroform, which completely removes any interference by the common excipients found in formulations.

Table 4 -Interference study

Sl. No	Excipients	Tolerance limit (μg ml¹)
1	Microcrystalline cellulose	94
2	Starch	168
3	Lactose	132
4	Povidone	64
5	Silicon dioxide	88
6	Titanium dioxide	58

Validation of the proposed method:

All the four proposed methods have been validated in terms of guideline proposed by International Conference on Harmonization²⁷ viz. selectivity, specificity, accuracy, precision, limits of calibration curve, LOD, LOQ, robustness, ruggedness and regression equation. The student t-test and variance F-test have been performed in comparison with a reference method. Table 1 summarizes the values for Beer’s law limits, molar absorptivity, regression equation, correlation coefficients, relative standard deviation and recoveries. To test the reproducibility of the proposed methods, six replicate determinations of 10µg ml^-1of Alprazolam were made. The coefficient of variation was found to be less than 1.2% for all the procedures.

The proposed methods have been successfully applied to the determination of Alprazolam in pharmaceutical preparations. The performance order of the proposed methods is BCG>BPB>BTB. The results obtained and shown in Table 2 and Table 3 were compared to those obtained by a reference method²⁷ by means of t-test at 95% confidence level. In all cases, the average results obtained by proposed methods and reference method were statistically identical, as the difference between the average values had no significance at 95% confidence level.

CONCLUSION:

In conclusion, Alprazolam forms ion-pair complexes with acidic triphenylmethane dyes viz., bromothymol blue, bromophenol blue and bromocresol green in 1:1 proportion. These complexes are extractable into chloroform and offer a basis for assay of the drug. The developed methods are simple, sensitive, reproducible and can be used for routine analysis of Alprazolam in pure and formulation forms.

ACKNOWLEDGEMENTS:

The authors are grateful to Prof. G. Venkateshwarlu, Department of Chemistry, Osmania University, Hyderabad for helpful discussion and to Sri M. Ravindra Reddy, Chairman, Managing Committee SAP College, Vikarabad for providing facilities. The authors are thankful to the UGC for financial assistance under Major Research Project.

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Received on 30.04.2020 Modified on 27.01.2022

Asian J. Research Chem. 2022; 15(4):259-264.

DOI: 10.52711/0974-4150.2022.00047